1. Field of the Invention
This invention pertains to the use of recombinant DNA methods to screen plants previously transformed with exogenous nucleic acids to determine whether the plants exhibit post-transcriptional gene silencing. More particularly, the invention relates to a rapid, simple method for screening large populations of transgenic plants, as well as for quantitating extent of suppression.
2. Background
Introduction of transgenes into plants can result in reduced expression of endogenous genes. Many plants exhibit such reduced expression as a result of post-transcriptional reduction of gene expression, a phenomenon that is referred to as post-transcriptional gene silencing (PTGS; for review, see, e.g., Baulcombe (1996) Plant Mol. Biol. 32: 79-88; Stam et al. (1997) Annals of Botany 79: 3-12). PTGS has been reported to involve degradation of nucleic acids that are related to the introduced transgene sequence. For example, the transcriptional rate of the endogenous gene is not altered in plants exhibiting sense suppression of that gene, but the accumulation of the mature mRNA for the endogenous gene is reduced. Elmayan and Vaucheret (1996) Plant J. 9: 787-797.
PTGS can have various effects. If one is attempting to obtain high level expression of a transgene, for example, PTGS of the transgene is counterproductive because it results in decreased expression of the transgene. Often, however, PTGS provides a convenient mechanism for altering the phenotype of a plant by reducing or eliminating the expression of a particular endogenous gene or transgene. PTGS can also mediate plant resistance to infection by viruses. Introduction of a transgene derived from a plant virus can induce post-transcriptional silencing of the corresponding gene of subsequently introduced virus, which can prevent establishment of viral infection. See, e.g., English et al. (1996) Plant Cell 8: 179-188.
The frequency with which post-transcriptional gene silencing is obtained in a population of plants, each of which is the result of an independent transformation event, can range widely, from less than 1% to 30% or more. A screening step is therefore useful in the production of plants which exhibit post-transcriptional gene silencing. Several screening methods have been used to select from a transgenic plant population those plants in which expression of a targeted gene is suppressed. These screening methods include:
1) Visual screening of a suitable trait (e.g., flower color);
2) Quantitation of the final product of a biosynthetic pathway that includes the protein product of the targeted gene as a pathway enzyme;
3) Quantitation of the protein product of the target gene;
4) Quantitation of the mRNA product of the target gene, using Northern analysis, RNase protection assay, RT-PCR, or other suitable technique;
5) Quantitation of the transgene mRNA in vegetative tissue using Northern analysis or other suitable technique.
Such methods have been used to screen transgenic populations for both anti-sense suppression and sense-mediated suppression. The first four of the above screening procedures require that the appropriate tissue of the mature plant is produced, and that extracts of the plant tissue are made for analysis. A significant drawback to these methods is that an appropriate tissue is sometimes produced only after an extended period of plant development, e.g., in flowers or fruit. In addition, the screening methods require one to prepare tissue extracts and perform complex molecular or biochemical analysis. The fifth screening method, quantitation of transgene mRNA in vegetative tissue, also has significant drawbacks. Theoretically, plants that exhibit only a small amount of transgene message in leaf tissue would be candidates for suppression of the endogenous gene. However, post-transcriptional silencing is but one of several mechanisms that could account for poor transgene expression. For example, poor transgene expression can result from insertion of the transgene in a transcriptionally inactive region of the genome.
Thus, a need exists for improved methods to screen for post-transcriptional silencing of gene expression. Preferably, these screening protocols would be suitable for analysis of young plants and plant tissues, and would not require complex procedures. Also needed are assays that can distinguish post-transcriptional silencing and other causes of poor transgene expression. The present invention fulfills these and other needs.
In a first embodiment, the invention provides a method for detecting post-transcriptional gene silencing (PTGS) in a plant cell. The method involves introducing into the plant cell a nucleic acid comprising a promoter operably linked to a suppression-sensitive reporter (SSR) gene which comprises a) a reporter coding sequence, and b) a targeting nucleotide sequence. The targeting nucleotide sequence is at least substantially identical to a region of a chosen gene. The reporter coding sequence and the targeting nucleotide sequence are transcribed as a single mRNA transcript. The level of expression of the SSR gene is determined to ascertain whether post-transcriptional gene silencing has occurred.
The invention also provides a method for detecting PTGS that involves, in addition to the use of an SSR gene, introducing into the plant cell a non-suppression sensitive reporter (NSR) gene. The NSR gene has a second reporter coding sequence which is different from the reporter coding sequence included in the SSR gene, and lacks a targeting nucleotide sequence. The level of expression of both the SSR gene and the NSR gene are determined. By comparing the expression levels, one can quantitate the degree of PTGS.
In another embodiment, the invention provides methods for detecting transgene-induced post-transcriptional silencing of a transgene in a plant cell by introducing into the plant cell a nucleic acid comprising a promoter operably linked to a suppression-sensitive reporter (SSR) gene which comprises a) a reporter coding sequence, and b) a targeting nucleotide sequence. The targeting nucleotide sequence is substantially identical to a region of the transgene, and is transcribed as a single mRNA transcript with the reporter coding sequence. The level of expression of the SSR gene is determined to ascertain whether post-transcriptional suppression has occurred.
A method for detecting transgene-induced post-transcriptional silencing of an endogerious gene in a plant cell is also provided by the invention. The method involves the steps of: 1) introducing into the plant cell a nucleic acid comprising a promoter operably linked to a suppression-sensitive reporter (SSR) gene which comprises a) a reporter coding sequence, and b) a targeting nucleotide sequence that is substantially identical to a region of the endogenous gene, wherein the reporter coding sequence and the targeting nucleotide sequence are transcribed as a single mRNA transcript; and 2) determining the level of expression of the SSR gene to determine whether post-transcriptional suppression has occurred.
The invention also provides methods for assaying a plant to determine whether a chosen gene is post-transcriptionally silenced. These methods involve introducing into cells obtained from the plant a nucleic acid comprising a promoter operably linked to a suppression-sensitive reporter (SSR) gene which comprises a) a reporter coding sequence, and b) a targeting nucleotide sequence that is substantially identical to a region of the chosen gene. The reporter coding sequence and the targeting nucleotide sequence are transcribed as a single mRNA transcript. A decrease in the level of expression of the SSR gene is indicative of post-transcriptional gene silencing.
Methods of identifying plant cells that are resistant to infection by a plant virus are also provided. These methods involve the steps of: 1) introducing into the plant cells a nucleic acid comprising a promoter operably linked to a suppression-sensitive reporter (SSR) gene which comprises a) a reporter coding sequence, and b) a targeting nucleotide sequence wherein such targeting nucleotide sequence is substantially identical to a region of a nucleotide sequence obtained from a plant virus, wherein the reporter coding sequence and the targeting nucleotide sequence are transcribed as a single mRNA transcript; and 2) determining the level of expression of the SSR gene. A reduction in SSR gene expression is indicative of cells that are resistant to infection by the plant virus.
The invention also provides plants in which expression of a chosen gene is post-transcriptionally suppressed, and parts obtained from such plants. The plants are obtained by culturing plant cells which have been determined to exhibit post-transcriptional suppression of the chosen gene by determining the level of expression of a suppression-sensitive reporter (SSR) gene which comprises a) a reporter coding sequence, and b) a targeting nucleotide sequence that is substantially identical to the chosen gene, wherein the reporter coding sequence and the targeting nucleotide sequence are transcribed as a single mRNA transcript. These plants can also include a transgene that is substantially identical to, or capable of specifically hybridizing to, the chosen gene.